*Target (protein/gene name):
Protein: NUCLEOSIDE TRIPHOSPATE DIPHOSPHOHYDROLASE 3
Gene: NTP3 *NCBI Gene # or RefSeq#: 7901558 *Protein ID (NP or XP #) or Wolbachia#: XP_002370463.1 *Organism (including strain):Toxoplasma gondii Etiologic Risk Group (see link below):Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents */Disease Information (sort of like the Intro to your Mini Research Write up):Toxoplasma gondii infects humans through meat, by ingestion of materials that had made contact with infected cat feces, and from infected pregnant mothers to their fetuses. This protozoan species causes the disease toxoplasmosis, which usually produces mild flu-like symptoms but tends to be more severe in people with weaker immune systems, such as infants and adults with HIV/AIDS. Link to TDR Targets page (if present): Not Available Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):http://toxodb.org/toxo/showRecord.do?name=GeneRecordClasses.GeneRecordClass&project_id=ToxoDB&source_id=TGGT1_360830 (searched through EuPath) Essentiality of this protein: "Via antisense RNA inhibition, it was demonstrated that NTPDase3 is required for replication of the parasite inside the host cells" [1].
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Multimer (4-mer) Complex of proteins?: Complexes with AMP, magnesium, and AMPPNP Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): "Pretreatment of parasites with a monoclonal antibody inhibiting both NTPDases significantly decreased the number of infected cells, suggesting that one or both isoforms are involved in invasion of the parasites into host cells" [1].
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): -- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3270962/ [1] -- links to assay reagents (substrates) pages:http://www.sigmaaldrich.com/catalog/product/sigma/flaas?lang=en®ion=US --- List cost and quantity of substrate reagents, supplier, and catalog #: $39.20 for 1 mg ATP, supplied by FLAAS SIGMA (SKU: FLAAS-1VL)
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 4A57
-- For Homology Model option: N/A
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors: Monoclonal antibodies Expression Information (has it been expressed in bacterial cells):Escherichia coli Rosetta pLysS [1] Purification Method: Exclusion chromatography using FPLC Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MTDSSSLRGVDADTEKRINVGKKHLQTLRNLETRCHDSLQALVVIDAGSSSTRTNVFLAKTRSCPNKGRSIDPDSIQLIG
KSIAGPDGKPLADLGWQVGVILHHVLFTEEWGRNAYEAGYSHNLEHHHHHH *length of your protein in Amino Acids: 611 amino acids Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 67.574 kilodaltons Molar Extinction coefficient of your protein at 280 nm wavelength: 52785 L/mol/cm (51910 L/mol/cm with Cys residues reduced) TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only):
CCACCTGTCCAGACGGCTAGGCGAAGACCCAGCTAGGTGCATGATTGATGAATACGGGGT
CTGGAAAGGACTGGGAGTCG *GC% Content for gene: 51.36% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
CCACCTGTCCAGACGGCTAGGCGAAGACCCAGCTAGGTGCATGATTGATGAATACGGGGT
CTGGAAAGGACTGGGAGTCG *GC% Content for gene (codon optimized): 51.36%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
References:
[1] Krug, U., Zebisch, M., Krauss, M., & Strater, N. (2012). Structural Insight into Activation Mechanism of Toxoplasma gondii Nucleoside Triphosphate Diphosphohydrolases by Disulfide Reduction. Journal of Biological Chemistry, 287(5), 3051-3066. Retrieved April 17, 2015, from NCBI.
Protein: NUCLEOSIDE TRIPHOSPATE DIPHOSPHOHYDROLASE 3
Gene: NTP3
*NCBI Gene # or RefSeq#: 7901558
*Protein ID (NP or XP #) or Wolbachia#: XP_002370463.1
*Organism (including strain): Toxoplasma gondii
Etiologic Risk Group (see link below): Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents
*/Disease Information (sort of like the Intro to your Mini Research Write up): Toxoplasma gondii infects humans through meat, by ingestion of materials that had made contact with infected cat feces, and from infected pregnant mothers to their fetuses. This protozoan species causes the disease toxoplasmosis, which usually produces mild flu-like symptoms but tends to be more severe in people with weaker immune systems, such as infants and adults with HIV/AIDS.
Link to TDR Targets page (if present): Not Available
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): http://toxodb.org/toxo/showRecord.do?name=GeneRecordClasses.GeneRecordClass&project_id=ToxoDB&source_id=TGGT1_360830 (searched through EuPath)
Essentiality of this protein: "Via antisense RNA inhibition, it was demonstrated that NTPDase3 is required for replication of the parasite inside the host cells" [1].
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Multimer (4-mer)
Complex of proteins?: Complexes with AMP, magnesium, and AMPPNP
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
"Pretreatment of parasites with a monoclonal antibody inhibiting both
NTPDases significantly decreased the number of infected cells, suggesting that
one or both isoforms are involved in invasion of the parasites into host cells" [1].
*EC#: 3.6.1.15
Link to BRENDA EC# page:
**http://www.brenda-enzymes.org/enzyme.php?ecno=3.6.1.15#PDB**
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3270962/ [1]
-- links to assay reagents (substrates) pages: http://www.sigmaaldrich.com/catalog/product/sigma/flaas?lang=en®ion=US
--- List cost and quantity of substrate reagents, supplier, and catalog #: $39.20 for 1 mg ATP, supplied by FLAAS SIGMA (SKU: FLAAS-1VL)
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 4A57
-- For Homology Model option: N/A
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives
---- Chain used for homology:
Current Inhibitors: Monoclonal antibodies
Expression Information (has it been expressed in bacterial cells): Escherichia coli Rosetta pLysS [1]
Purification Method: Exclusion chromatography using FPLC
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MTDSSSLRGVDADTEKRINVGKKHLQTLRNLETRCHDSLQALVVIDAGSSSTRTNVFLAKTRSCPNKGRSIDPDSIQLIG
AGKRFAGLRVVLEEWLDTYAGKDWESRPVDARLLFQYVPQMHEGAKKLMQLLEEDTVAILDSQLNEKQKVQVKALGIPVM
LCSTAGVRDFHEWYRDALFVLLRHLINNPSPAHGYKFFTNPFWTRPITGAEEGLFAFITLNHLSRRLGEDPARCMIDEYG
VKQCRNDLAGVVEVGGASAQIVFPLQEGTVLPSSVRAVNLQRERLLPERYPSADVVSVSFMQLGMASSAGLFLKELCSND
EFLQGGICSNPCLFKGFQQSCSAGEVEVRPDGSASVNEDVRKNRLKPLATYCSVNNPEISFKVTNEMQCRENSIDPTKPL
AERMKIENCSIIKGTGNFDKCVSQVESILVAPKLPLPANIEAASSGFESVDQVFRFASSTAPMIVTGGGMLAAINTLKDH
RLLRSDFSGDVEELAEAAREFCSSEVIIRTDGPVIQLPNARGEQKLNSLNFDLCKTMALTVSLLRHMAAGENQPSFIKWE
KSIAGPDGKPLADLGWQVGVILHHVLFTEEWGRNAYEAGYSHNLEHHHHHH
*length of your protein in Amino Acids: 611 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 67.574 kilodaltons
Molar Extinction coefficient of your protein at 280 nm wavelength: 52785 L/mol/cm (51910 L/mol/cm with Cys residues reduced)
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
CCACCTGTCCAGACGGCTAGGCGAAGACCCAGCTAGGTGCATGATTGATGAATACGGGGT
GAAGCATTGCCGCAATGACCTTGCTGGCGTGGTCGAGGTTGGGGGTGCTTCTGCTCAAAT
TGTCTTTCCACTACAGGAAGGTACCGTCCTGCCCTCTTCGGTCCGTGCCGTCAACCTGCA
GCGCGAACGCCTTCTCCCAGAACGTTATCCAAGTGCCGACGTCGTGTCAGTTTCGTTTAT
GCAGCTGGGTATGGCCAGTTCCGCAGGACTGTTTCTCAAGGAGCTGTGTAGTAACGATGA
GTTTTTGCAAGGCGGAATTTGCTCCAACCCGTGTTTATTTAAGGGCTTTCAGCAGAGTTG
TTCCGCGGGCGAAGTCGAGGTCCGTCCTGACGGATCCGCATCTGTGAACGAAGATGTGAG
AAAGAACAGGCTGAAGCCGCTTGCAACGTACTGCTCAGTACATAACCCGGAAATCAGCTT
CAAGGTAACCAACGAAATGCAGTGCCGGGAAAACTCGATTGATCCAACGAAGCCACTCGC
GGAGAGAATGAAGATCGAGAACTGCAGTATAATCGAAGGAACCGGAAACTTTGACAAATG
CGTGAGCCAGGTAGAAAGCATTCTCGTCGCTCCCAAGTTACCTCTACCAGCCAACATTGA
AGCTGCGTCCTCGGGCTTCGAATCCGTCGACCAAGTCTTCAGGTTTGCCTCCTCGACAGC
ACCAATGTTTATAACGGGACGAGAGATGCTTGCGTCTATAGACACACTCAAAGATCACCG
TCTTCTTCGGAGCGACTTCAGCGGTGACGTAGAAGAACTGGCGGAAGCGGCGCGTGAATT
CTGTTCTTCTGAAGTGATTATCCGTACAGATGGCCCGGTCATCCAGTTGCCAAACGCGCG
AGGGGAACAGAAGCTGAATAGCCTAAACTTCGACTTGTGCAAGACGATGGCGCTGACTGT
GTCTCTGCTTAGGCACATGGCGGCAGGAGAAAATCAACCGTCGTTCATTAAATGGGAAAA
AAGCATTGCAGGACCTGATGGGAAACCACTTGCTGACCTTGGGTGGCAAGTAGGAGTCAT
TCTTCACCACGTCCTCTTCACTGAAGAATGGGGACGTAACGCTTATGAGGCGGGATATTC
TCACAATCTGTGAAATGTCGATTGATGGTGTCGCCTATTATCGGAGAAGATCGTGCTTGC
TACACCAGTGTCTTTCATACTCCTGTGTACTATAGTTCAGATGAGTCGAGGGCTTGCGGA
GAGGTAGTTCTCTAGTGGTGATACGCCGGGGATCTGACCTTTTTGTTGTCGGCTATCGTG
AGACCTGTCTTCCGCGAGCAAGCGCCTTACGTTGTCCTTCGAAGCGCTCCTCGATGGAGC
AAATTCATGCGGGATTTATACCAATAGGGCCGACTCGGAAGAAGATCGGACACCCATGTT
TTTACGCTGCACTTGCGATAACGTAACAACCCGGTAGGCTGCTTATCTCAACTGTTTTAT
TACAGGTTACCGTGTTCGTCTACAGTCCCCAGAAACGCGATGCGTTCCTCTCATCGTGCT
CGTCTCAAGACGACCATCACAAGCACGTGCGCGCTCTAATGTGGTGACAATGTGACGCCG
CTCAATTCTCGCTTATGCTCTGCTCGTTTGTATTGTGTGTTGAAACTGAGCTGCGAACAA
CATTTGATGGCTCCGTGCATTAGCCGGCGCCTCAAGGATTCCTGTGTGATGGACGCCAGT
GACACGCCGACGTCTGTTTTTTGCATGCTCGGCCGTTCGTCCGGTTAGAACTGACAGCAT
GCCTGGTAACAATATAATGGCTGGAAGCGTTGAAACGGTCCTCACGGACTTCACGCACGG
TGTCGCGCCAGAATTGCGTTTCTGATTTCAGCAGCGTTTTTGCACACTTGCAGCGGCAAG
AAGCAACTCAGAAAAGGCTGGTCAATCTATGTAGGTTAGGCACACAGCCGCATCACTGTC
CTTTACATCATTAATTACATGTCTTGTCCCATCTTTTATCATATTCAATCTATATAGGGC
AGCATGAGCAGAGAAGACGCATAGGAGTCTGTCTTTCCGACGGTGTAAGAGGAATAATGG
TGTAAAGTCGTTCTTTTCTACCCGACCAGTGTTACCTCGTTATCTAAAACTGAGTCCTGA
AAAGGCCCACATTCATCGACAAGAAAGGTCCCTGGCCATCAAACAGCAAAGGAGGGAACA
AAGAACGGGGACGCACAGTTCTACAGAGAGGGAATCGAATGCTTTTTTACTTTCGAGTTG
CGGCAGTGAGGACAATGCTTGTCCCGACTGTGCATGCAGACCGTGGCGCCACACTAGTGT
AGGCCTCAGCGGCTGACGCACAAGATCCATTTACCACAGAAACGGAGGTGCTCGAGACCA
GAATGACTTCTCTTGCGGATGTCGTTGGACGATGGAAATCGTGAGGAAGGGGAGGAAACG
AGAGACGCGAAGAGGGCGCAACCCGATTTCGGAGAGGACCGGAAGTGCGTGCCGACCACC
AGGGCGTCTAGCTCAAATGGTAGAGCGCTCGCTTAGCATGCGAGAGGTAATGGGATCGAT
ACCCATGTCGTCCAGTTCTTTTCCGAAATTCCAACTCATTGCGACACTTTTAAACTGCTT
CGATGTTCCGAAAATGTCCGGCCTCTCATCTGATACGTCTTGCTGACACGGGCCGAAATA
GAAAACTCTACTTGCTCTCTGTGGCTGCGTCATATTGCGCCATATCGTGTGTACGGCAAC
GGAGTCAGCCGGATGAACAGCTGCTGTTGACGATTCTGTCCTATTTTCAATTCACGATGA
AAATACTATGAAAAATAGTATGTAGACGTTTTGCTGTAAATGAAGAAATATGCAGAGAAA
CTCGTATCACATGTCGCTGCAAGCCGCCACTAGATCGTCCACAAACCTGTTCCGCTCAGT
TGGCTTTCGTACCATCCGGGCAAGCGCACACAACGGGTCTAGACCGCAGCCTCCTGACTT
CACAAATCTCGAAGTAAGCAGGCTCACTCCTAATTTTCCTCAAGAACCATAAGCATCTAC
CGCCGAGCTGCAACAGTGAGCTCGCCTGTATTCCTCAACCCGCCCTACGACGTCACCCCC
GCGACCGCGGAAATCTGGGTTTTGGATCACGAATCAGTTGACCTGTCCTCCGCTCTAGAG
TCCACTGCATTGAACCGGGAACATTTTTATCGGTTCTACAAGTTTGGAATACGCATGTGT
CTGTTCCCACACCAAGTAACTGCGTCCGTCTGGTCTGTTTCAATTATGTAACTTGGCAAC
GTTGTCGCGATGGCCCTCGCCCGTAATCTGCTCGGATATCACACTGACCATGCCACTTCA
CTTATTTGAGCACATGAAACGGTCGGTGGATGAGCAGTTCAGGACAAACATGCTTGTCGC
TGTCTCAGTCTCTCCAGTTGTTCGGTGTTCTAGGGTGAGTGACGCTGCAGCCAAGCCAGC
CGTAACTCGCGTCTCAAAGGGCACCGGGGCCAGAGTTTGGCAGCCACTTTTTCACACGGC
ACTTCGGCGCACATTTCAGGGTGCACGCGCCAGGCACAGCCGACCGCTTCACGAGGTGTT
AGTGACGCATACCTCGTTCGCGTCTCTCTTCGAATACGGGACACAAACAAATTCCGTTCA
CGACAGATCACCGAAGCGTCATTGGGCACTCATTGTTAACAAACTGCCGTAGCCGATCCG
TTCGCACGTCATACTCGGCCTCGAGCACCGCGCACGTCCTACCCGGAGTCCTCATTGTAT
CCGATCCCGGTACCTCTTTCATTCTACCGCAGCTTCCAGTGTACTTTCCGCGTCACCAAA
GCGGTCAGTAGAACCAGGTTCGAGCTGCTGTTCAGTTTTTTTGACGATCGACGCGTCTTG
CCTTTACTGTTGTGTTCGTACGAATCAGCTGCCGTTTATAGGTAGGCATCATGTGGTTGC
CGGTGTATGTGCCTCTCTTGCTGGTGTTTGGAGTTAGTTTGTCCCTCCCACAAGGAAGCC
TTGGCACAGACTCATCGTCACTCCGGGGAGTGGACGCTGACACTGAAAAGCGGATTAATG
TCGGGAAGAAACACCTGCAGACCCTCCGAAATCTGGAAACTCGGTGCCACGATTCCTTGC
AAGCACTTGTTGTAATCGATGCAGGCAGTAGTTCGACGAGGACGAATGTTTTCTTGGCGA
AAACCCGGTCATGCCCAAATAAAGGCCGTAGCATTGACCCAGACAGTATTCAACTCATCG
GCGCTGGCAAGCGGTTCGCAGGACTTCGTGTAGTTTTAGAAGAGTGGCTGGACACTTACG
CTGGAAAGGACTGGGAGTCG
*GC% Content for gene: 51.36%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
CCACCTGTCCAGACGGCTAGGCGAAGACCCAGCTAGGTGCATGATTGATGAATACGGGGT
GAAGCATTGCCGCAATGACCTTGCTGGCGTGGTCGAGGTTGGGGGTGCTTCTGCTCAAAT
TGTCTTTCCACTACAGGAAGGTACCGTCCTGCCCTCTTCGGTCCGTGCCGTCAACCTGCA
GCGCGAACGCCTTCTCCCAGAACGTTATCCAAGTGCCGACGTCGTGTCAGTTTCGTTTAT
GCAGCTGGGTATGGCCAGTTCCGCAGGACTGTTTCTCAAGGAGCTGTGTAGTAACGATGA
GTTTTTGCAAGGCGGAATTTGCTCCAACCCGTGTTTATTTAAGGGCTTTCAGCAGAGTTG
TTCCGCGGGCGAAGTCGAGGTCCGTCCTGACGGATCCGCATCTGTGAACGAAGATGTGAG
AAAGAACAGGCTGAAGCCGCTTGCAACGTACTGCTCAGTACATAACCCGGAAATCAGCTT
CAAGGTAACCAACGAAATGCAGTGCCGGGAAAACTCGATTGATCCAACGAAGCCACTCGC
GGAGAGAATGAAGATCGAGAACTGCAGTATAATCGAAGGAACCGGAAACTTTGACAAATG
CGTGAGCCAGGTAGAAAGCATTCTCGTCGCTCCCAAGTTACCTCTACCAGCCAACATTGA
AGCTGCGTCCTCGGGCTTCGAATCCGTCGACCAAGTCTTCAGGTTTGCCTCCTCGACAGC
ACCAATGTTTATAACGGGACGAGAGATGCTTGCGTCTATAGACACACTCAAAGATCACCG
TCTTCTTCGGAGCGACTTCAGCGGTGACGTAGAAGAACTGGCGGAAGCGGCGCGTGAATT
CTGTTCTTCTGAAGTGATTATCCGTACAGATGGCCCGGTCATCCAGTTGCCAAACGCGCG
AGGGGAACAGAAGCTGAATAGCCTAAACTTCGACTTGTGCAAGACGATGGCGCTGACTGT
GTCTCTGCTTAGGCACATGGCGGCAGGAGAAAATCAACCGTCGTTCATTAAATGGGAAAA
AAGCATTGCAGGACCTGATGGGAAACCACTTGCTGACCTTGGGTGGCAAGTAGGAGTCAT
TCTTCACCACGTCCTCTTCACTGAAGAATGGGGACGTAACGCTTATGAGGCGGGATATTC
TCACAATCTGTGAAATGTCGATTGATGGTGTCGCCTATTATCGGAGAAGATCGTGCTTGC
TACACCAGTGTCTTTCATACTCCTGTGTACTATAGTTCAGATGAGTCGAGGGCTTGCGGA
GAGGTAGTTCTCTAGTGGTGATACGCCGGGGATCTGACCTTTTTGTTGTCGGCTATCGTG
AGACCTGTCTTCCGCGAGCAAGCGCCTTACGTTGTCCTTCGAAGCGCTCCTCGATGGAGC
AAATTCATGCGGGATTTATACCAATAGGGCCGACTCGGAAGAAGATCGGACACCCATGTT
TTTACGCTGCACTTGCGATAACGTAACAACCCGGTAGGCTGCTTATCTCAACTGTTTTAT
TACAGGTTACCGTGTTCGTCTACAGTCCCCAGAAACGCGATGCGTTCCTCTCATCGTGCT
CGTCTCAAGACGACCATCACAAGCACGTGCGCGCTCTAATGTGGTGACAATGTGACGCCG
CTCAATTCTCGCTTATGCTCTGCTCGTTTGTATTGTGTGTTGAAACTGAGCTGCGAACAA
CATTTGATGGCTCCGTGCATTAGCCGGCGCCTCAAGGATTCCTGTGTGATGGACGCCAGT
GACACGCCGACGTCTGTTTTTTGCATGCTCGGCCGTTCGTCCGGTTAGAACTGACAGCAT
GCCTGGTAACAATATAATGGCTGGAAGCGTTGAAACGGTCCTCACGGACTTCACGCACGG
TGTCGCGCCAGAATTGCGTTTCTGATTTCAGCAGCGTTTTTGCACACTTGCAGCGGCAAG
AAGCAACTCAGAAAAGGCTGGTCAATCTATGTAGGTTAGGCACACAGCCGCATCACTGTC
CTTTACATCATTAATTACATGTCTTGTCCCATCTTTTATCATATTCAATCTATATAGGGC
AGCATGAGCAGAGAAGACGCATAGGAGTCTGTCTTTCCGACGGTGTAAGAGGAATAATGG
TGTAAAGTCGTTCTTTTCTACCCGACCAGTGTTACCTCGTTATCTAAAACTGAGTCCTGA
AAAGGCCCACATTCATCGACAAGAAAGGTCCCTGGCCATCAAACAGCAAAGGAGGGAACA
AAGAACGGGGACGCACAGTTCTACAGAGAGGGAATCGAATGCTTTTTTACTTTCGAGTTG
CGGCAGTGAGGACAATGCTTGTCCCGACTGTGCATGCAGACCGTGGCGCCACACTAGTGT
AGGCCTCAGCGGCTGACGCACAAGATCCATTTACCACAGAAACGGAGGTGCTCGAGACCA
GAATGACTTCTCTTGCGGATGTCGTTGGACGATGGAAATCGTGAGGAAGGGGAGGAAACG
AGAGACGCGAAGAGGGCGCAACCCGATTTCGGAGAGGACCGGAAGTGCGTGCCGACCACC
AGGGCGTCTAGCTCAAATGGTAGAGCGCTCGCTTAGCATGCGAGAGGTAATGGGATCGAT
ACCCATGTCGTCCAGTTCTTTTCCGAAATTCCAACTCATTGCGACACTTTTAAACTGCTT
CGATGTTCCGAAAATGTCCGGCCTCTCATCTGATACGTCTTGCTGACACGGGCCGAAATA
GAAAACTCTACTTGCTCTCTGTGGCTGCGTCATATTGCGCCATATCGTGTGTACGGCAAC
GGAGTCAGCCGGATGAACAGCTGCTGTTGACGATTCTGTCCTATTTTCAATTCACGATGA
AAATACTATGAAAAATAGTATGTAGACGTTTTGCTGTAAATGAAGAAATATGCAGAGAAA
CTCGTATCACATGTCGCTGCAAGCCGCCACTAGATCGTCCACAAACCTGTTCCGCTCAGT
TGGCTTTCGTACCATCCGGGCAAGCGCACACAACGGGTCTAGACCGCAGCCTCCTGACTT
CACAAATCTCGAAGTAAGCAGGCTCACTCCTAATTTTCCTCAAGAACCATAAGCATCTAC
CGCCGAGCTGCAACAGTGAGCTCGCCTGTATTCCTCAACCCGCCCTACGACGTCACCCCC
GCGACCGCGGAAATCTGGGTTTTGGATCACGAATCAGTTGACCTGTCCTCCGCTCTAGAG
TCCACTGCATTGAACCGGGAACATTTTTATCGGTTCTACAAGTTTGGAATACGCATGTGT
CTGTTCCCACACCAAGTAACTGCGTCCGTCTGGTCTGTTTCAATTATGTAACTTGGCAAC
GTTGTCGCGATGGCCCTCGCCCGTAATCTGCTCGGATATCACACTGACCATGCCACTTCA
CTTATTTGAGCACATGAAACGGTCGGTGGATGAGCAGTTCAGGACAAACATGCTTGTCGC
TGTCTCAGTCTCTCCAGTTGTTCGGTGTTCTAGGGTGAGTGACGCTGCAGCCAAGCCAGC
CGTAACTCGCGTCTCAAAGGGCACCGGGGCCAGAGTTTGGCAGCCACTTTTTCACACGGC
ACTTCGGCGCACATTTCAGGGTGCACGCGCCAGGCACAGCCGACCGCTTCACGAGGTGTT
AGTGACGCATACCTCGTTCGCGTCTCTCTTCGAATACGGGACACAAACAAATTCCGTTCA
CGACAGATCACCGAAGCGTCATTGGGCACTCATTGTTAACAAACTGCCGTAGCCGATCCG
TTCGCACGTCATACTCGGCCTCGAGCACCGCGCACGTCCTACCCGGAGTCCTCATTGTAT
CCGATCCCGGTACCTCTTTCATTCTACCGCAGCTTCCAGTGTACTTTCCGCGTCACCAAA
GCGGTCAGTAGAACCAGGTTCGAGCTGCTGTTCAGTTTTTTTGACGATCGACGCGTCTTG
CCTTTACTGTTGTGTTCGTACGAATCAGCTGCCGTTTATAGGTAGGCATCATGTGGTTGC
CGGTGTATGTGCCTCTCTTGCTGGTGTTTGGAGTTAGTTTGTCCCTCCCACAAGGAAGCC
TTGGCACAGACTCATCGTCACTCCGGGGAGTGGACGCTGACACTGAAAAGCGGATTAATG
TCGGGAAGAAACACCTGCAGACCCTCCGAAATCTGGAAACTCGGTGCCACGATTCCTTGC
AAGCACTTGTTGTAATCGATGCAGGCAGTAGTTCGACGAGGACGAATGTTTTCTTGGCGA
AAACCCGGTCATGCCCAAATAAAGGCCGTAGCATTGACCCAGACAGTATTCAACTCATCG
GCGCTGGCAAGCGGTTCGCAGGACTTCGTGTAGTTTTAGAAGAGTGGCTGGACACTTACG
CTGGAAAGGACTGGGAGTCG
*GC% Content for gene (codon optimized): 51.36%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
References:
[1] Krug, U., Zebisch, M., Krauss, M., & Strater, N. (2012). Structural Insight into Activation Mechanism of Toxoplasma gondii Nucleoside Triphosphate Diphosphohydrolases by Disulfide Reduction. Journal of Biological Chemistry, 287(5), 3051-3066. Retrieved April 17, 2015, from NCBI.